NOVEL POLYPEPTIDES AND MEDICAL USES THEREOF
20210403535 · 2021-12-30
Inventors
Cpc classification
C07K14/78
CHEMISTRY; METALLURGY
C07K14/4723
CHEMISTRY; METALLURGY
A61L29/048
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
A61L15/32
HUMAN NECESSITIES
A61L2420/00
HUMAN NECESSITIES
A61L29/16
HUMAN NECESSITIES
A61L31/16
HUMAN NECESSITIES
A61K9/0053
HUMAN NECESSITIES
A61L17/005
HUMAN NECESSITIES
A61L27/227
HUMAN NECESSITIES
A61L27/54
HUMAN NECESSITIES
A61K9/7023
HUMAN NECESSITIES
A61L24/108
HUMAN NECESSITIES
A61L31/047
HUMAN NECESSITIES
A61L2300/404
HUMAN NECESSITIES
A61K9/0019
HUMAN NECESSITIES
C07K14/755
CHEMISTRY; METALLURGY
A61K9/0014
HUMAN NECESSITIES
A61L2300/252
HUMAN NECESSITIES
International classification
C07K14/78
CHEMISTRY; METALLURGY
A61L15/32
HUMAN NECESSITIES
A61L17/00
HUMAN NECESSITIES
A61L24/00
HUMAN NECESSITIES
A61L27/22
HUMAN NECESSITIES
A61L27/54
HUMAN NECESSITIES
A61L29/16
HUMAN NECESSITIES
A61L31/16
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
Abstract
The present invention provides polypeptides comprising or consisting of an amino acid sequence derived from collagen type VI or a fragment, variant, fusion or derivative thereof, or a fusion of said fragment, variant of derivative thereof, wherein the polypeptide, fragment, variant, fusion or derivative is capable of killing or attenuating the growth of microorganisms. Related aspects of the invention provide corresponding isolated nucleic acid molecules, vectors and host cells for making the same. Additionally provided are pharmaceutical compositions comprising a polypeptide of the invention, as well as methods of use of the same in the treatment and/or prevention of microbial infections and in wound care. Also provided are a method of killing microorganisms in vitro and a medical device associated with the pharmaceutical composition.
Claims
1. A polypeptide consisting of an amino acid sequence derived from the α3 chain of collagen type VI, or a fragment, variant, fusion or derivative thereof, or a fusion of said fragment, variant of derivative thereof, wherein the polypeptide, fragment, variant, fusion or derivative is capable of killing or attenuating the growth of microorganisms.
2. A polypeptide according to claim 1 wherein the microorganisms are selected from the group consisting of bacteria, mycoplasmas, yeasts, fungi and viruses.
3. A polypeptide according to any one of the preceding claims wherein the polypeptide is capable of binding to the membrane of the microorganism.
4. A polypeptide according to any one of the preceding claims wherein the polypeptide is capable of causing membrane disruption of the microorganisms.
5. A polypeptide according to any one of the preceding claims which is capable of promoting wound closure.
6. A polypeptide according to any one of the preceding claims, wherein the polypeptide is capable of exhibiting an antimicrobial effect greater than or equal to that of LL-37.
7. A polypeptide according to any one of the preceding claims wherein the microorganisms are Gram-positive or Gram-negative bacteria.
8. A polypeptide according to claim 7, wherein the microorganisms are selected from the group consisting of: Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, group A streptococcus (e.g. Streptococcus pyogenes), group B streptococcus (e.g. Streptococcus agalactiae), group C streptococcus (e.g. Streptococcus dysgalactiae), group D streptococcus (e.g. Enterococcus faecalis), group F streptococcus (e.g. Streptococcus anginosus), group G streptococcus (e.g. Streptococcus dysgalactiae equisimilis), alpha-hemolytic streptococcus (e.g. Streptococcus viridans, Streptococcus pneumoniae), Streptococcus bovis, Streptococcus mitis, Streptococcus anginosus, Streptococcus sanguinis, Streptococcus suis, Streptococcus mutans, Moraxella catarrhalis, Non-typeable Haemophilus influenzae (NTHi), Haemophilus influenzae b (Hib), Actinomyces naeslundii, Fusobacterium nucleatum, Prevotella intermedia, Klebsiella pneumoniae, Enterococcus cloacae, Enterococcus faecalis, Staphylococcus epidermidis, multi-resistant Pseudomonas aeruginosa (MRPA), multi-resistant Staphylococcus aureus (MRSA), multi-resistant Escherichia coli (MREC), multi-resistant Staphylococcus epidermidis (MRSE) and multi-resistant Klebsiella pneumoniae (MRKP).
9. A polypeptide according to any one of the preceding claims wherein the microorganisms are bacteria which are resistant to one or more conventional antibiotic agents.
10. A polypeptide according to claim 9 wherein the microorganism is selected from the group consisting of: multidrug-resistant Staphylococcus aureus (M RSA), multidrug-resistant Pseudomonas aeruginosa (MRPA), multi-resistant Escherichia coli (MREC), multi-resistant Staphylococcus epidermidis (MRSE) and multi-resistant Klebsiella pneumoniae (MRKP).
11. A polypeptide according to any one of the preceding claims wherein the polypeptide is substantially non-toxic to mammalian cells.
12. A polypeptide according to any one of the preceding claims wherein the polypeptide is capable of exerting an anti-endotoxic effect.
13. A polypeptide according to any one of the preceding claims wherein the polypeptide is derived from a von Willebrand Factor type A domain.
14. A polypeptide according to any of the preceding claims wherein the polypeptide is a fragment of the α3 chain of collagen type VI.
15. A polypeptide according to any one of the preceding claims wherein the polypeptide is derived from the N2, N3 or C1 domain of the α3 chain of collagen type VI.
16. A polypeptide according to any one of the preceding claims wherein the polypeptide has a net positive charge.
17. A polypeptide according to claim 16 wherein the charge on the polypeptide ranges from between +2 to +9.
18. A polypeptide according to any one of the preceding claims wherein the polypeptide has at least 30% hydrophobic residues.
19. A polypeptide according to any one of the preceding claims comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 23, and fragments, variants, fusions or derivatives thereof, and fusions of said fragments, variants and derivatives thereof, which retain an antimicrobial activity of any one of SEQ ID NOs:1 to 23.
20. A polypeptide according to claim 19 comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5: TABLE-US-00006 “GVR28”: [SEQ ID NO: 1] GVRPDGFAHIRDFVSRIVRRLNIGPSKV “FYL25”: [SEQ ID NO: 2] FYLKTYRSQAPVLDAIRRLRLRGGS “FFL25”: [SEQ ID NO: 3] FFLKDFSTKRQIIDAINKVVYKGGR “VTT30”: [SEQ ID NO: 4] VTTEIRFADSKRKSVLLDKIKNLQVALTSK “SFV33”: [SEQ ID NO: 5] SFVARNTFKRVRNGFLMRKVAVFFSNTPTRASP and fragments, variants, fusions or derivatives thereof, and fusions of said fragments, variants and derivatives thereof, which retain an antimicrobial activity of any one of SEQ ID NOs:1 to 5.
21. A polypeptide according to claim 20 comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5.
22. A polypeptide according to any one of the preceding claims wherein the polypeptide comprises or consists of a variant of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 23.
23. A polypeptide according to claim 22 wherein the variant has at least 50% identity with the amino acid sequence amino acid sequence of any one of SEQ ID NOs: 1 to 23, for example at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity.
24. A polypeptide according to any one of the preceding claims wherein the polypeptide is between 10 and 200 amino acids in length, for example between 10 and 150, 15 and 100, 15 and 50, 20 and 40 or 25 and 35 amino acids in length.
25. A polypeptide according to claim 24 wherein the polypeptide is at least 20 amino acids in length.
26. A polypeptide according to any one of the preceding claims wherein the polypeptide, or fragment, variant, fusion or derivative thereof, comprises one or more amino acids that are modified or derivatised.
27. A polypeptide according to claim 26 wherein the one or more amino acids are modified or derivatised by PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.
28. A polypeptide according to any one of the preceding claims wherein the polypeptide is a recombinant polypeptide.
29. A nucleic acid molecule which encodes a polypeptide according to any one of the preceding claims.
30. A vector comprising a nucleic acid molecule according to claim 29.
31. A vector according to claim 30 wherein the vector is an expression vector.
32. A host cell comprising a nucleic acid molecule according to claim 29 or a vector according to claim 30 or 31.
33. A method of making a polypeptide according to any one of claims 1 to 28 comprising culturing a population of host cells according to claim 32 under conditions in which said polypeptide is expressed, and isolating the polypeptide therefrom.
34. A method of making a polypeptide according to any one of claims 1 to 28 comprising liquid-phase or solid-phase synthesis of the polypeptide.
35. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 28 together with a pharmaceutically acceptable excipient, diluent, carrier, buffer or adjuvant.
36. A pharmaceutical composition according to claim 35 suitable for administration via a route selected from the group consisting of oral administration, parenteral administration and topical administration.
37. A pharmaceutical composition according to claim 36 suitable for topical administration.
38. A medical device, implant, wound care product, or material for use in the same, which is coated, impregnated, admixed or otherwise associated with a pharmaceutical composition according to any one of claims 35 to 37.
39. A medical device, implant, wound care product, or material for use in the same, according to claim 38 wherein the device, implant, wound care product, or material is for use in by-pass surgery, extracorporeal circulation, wound care and/or dialysis.
40. A medical device, implant, wound care product, or material for use in the same, according to claim 38 or 39 wherein the pharmaceutical composition is coated, painted, sprayed or otherwise applied to a suture, prosthesis, implant, wound dressing, catheter, lens, skin graft, skin substitute, fibrin glue or bandage.
41. A medical device, implant, wound care product, or material for use in the same, according to any one of claims 38 to 40 comprising or consisting of a polymer, metal, metal oxide and/or ceramic.
42. A kit comprising a pharmaceutical composition according to any one of claims 35 to 37 or a medical device, implant, wound care product, or material for use in the same, according to any one of claims 38 to 41.
43. A polypeptide as defined in any one of claims 1 to 28, or a nucleic acid molecule as defined in claim 29, or a pharmaceutical composition as defined in any one of claims 35 to 37 for use in medicine.
44. A polypeptide as defined in any one of claims 1 to 28, or a nucleic acid molecule as defined in claim 29, or a pharmaceutical composition as defined in any one of claims 35 to 37 for use in the curative and/or prophylactic treatment of microbial infections.
45. A polypeptide, nucleic acid molecule, or pharmaceutical composition for use according to claim 44 wherein the microbial infection is a systemic infection.
46. A polypeptide, nucleic acid molecule, or pharmaceutical composition for use according to claim 44 or 45 wherein the microbial infection is resistant to one or more conventional antibiotic agents.
47. A polypeptide, nucleic acid molecule, or pharmaceutical composition for use according to any one of claims 44 to 46 wherein the microbial infection is caused by a microorganism selected from the group consisting of: Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, group A streptococcus (e.g. Streptococcus pyogenes), group B streptococcus (e.g. Streptococcus agalactiae), group C streptococcus (e.g. Streptococcus dysgalactiae), group D streptococcus (e.g. Entero-coccus faecalis), group F streptococcus (e.g. Streptococcus anginosus), group G streptococcus (e.g. Streptococcus dysgalactiae equisimilis), alpha-hemolytic streptococcus (e.g. Streptococcus viridans, Streptococcus pneumoniae), Streptococcus bovis, Streptococcus mitis, Streptococcus anginosus, Streptococcus sanguinis, Streptococcus suis, Streptococcus mutans, Moraxella catarrhalis, Non-typeable Haemophilus influenzae (NTHi), Haemophilus influenzae b (Hib), Actinomyces naeslundii, Fusobacterium nucleatum, Prevotella intermedia, Klebsiella pneumoniae, Enterococcus cloacae, Enterococcus faecalis, Staphylococcus epidermidis, multi-resistant Pseudomonas aeruginosa (MRPA), and multi-resistant Staphylococcus aureus (MRSA), multi-resistant Escherichia coli (MREC), multi-resistant Staphylococcus epidermidis (MRSE) and multi-resistant Klebsiella pneumoniae (MRKP).
48. A polypeptide, nucleic acid molecule, or pharmaceutical composition for use according to any one of claims 44 to 47 wherein the microbial infection is caused by a microorganism selected from the group consisting of: multidrug-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MRPA).
49. A polypeptide, nucleic acid molecule, or pharmaceutical composition for use according to any one of claims 44 to 48 in combination with one or more additional antimicrobial agents.
50. A polypeptide, nucleic acid molecule, or pharmaceutical composition for use according to claim 49 wherein the one or more additional antimicrobial agent is selected from the group consisting of: antimicrobial polypeptides and antibiotics.
51. A polypeptide as defined in any one of claims 1 to 28, or a nucleic acid molecule as defined in claim 29, or a pharmaceutical composition as defined in any one of claims 35 to 37 for use in wound care.
52. Use of a polypeptide or fragment as defined in any one of claims 1 to 28, or a nucleic acid molecule as defined in claim 28, or a pharmaceutical composition as defined in any one of claims 35 to 37 in the manufacture of a medicament for the treatment of microbial infections.
53. Use of a peptide or fragment as defined in any one of claims 1 to 28, or a nucleic acid molecule as defined in claim 29, or a pharmaceutical composition as defined in any one of claims 35 to 37 in the manufacture of a medicament for the treatment of wounds.
54. A method of treating an individual with a microbial infection, the method comprising the step of administering to an individual in need thereof an effective amount of a peptide or fragment as defined in any of claims 1 to 28, or a nucleic acid molecule as defined in claim 29, or a pharmaceutical composition as defined in any one of claims 35 to 37.
55. A method of treating a wound in an individual, the method comprising the step of administering to an individual in need thereof an effective amount of a peptide or fragment as defined in any of claims 1 to 28, or a nucleic acid molecule as defined in claim 29, or a pharmaceutical composition as defined in any one of claims 35 to 37.
56. A method for killing microorganisms in vitro comprising contacting the microorganisms with a polypeptide as described in any of claims 1 to 28, or a nucleic acid molecule as defined in claim 29, or a pharmaceutical composition as defined in any one of claims 35 to 37.
Description
[0180] Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:
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EXAMPLES
Example A
[0198] Introduction
[0199] The purpose of this study was to investigate if the globular domains of collagen type VI have a role in host defence during infection, and if peptides derived from these domains have similar properties.
[0200] Materials and Methods
[0201] Bacterial Strains and Culture Conditions
[0202] Streptococcus pyogenes strain AP1 (40/58) of serotype M1 was from the World Health Organization Collaborating Centre for Reference and Research on Streptococci, Prague, Czech Republic. Staphylococcus aureus strain 111 and Escherichia coli strain B1351 were collected at the Department of Clinical Microbiology, Lund University Hospital, Sweden. The Pseudomonas aeruginosa strain used in this study was PAO1 (ATCC, Teddington Oly, UK), originally isolated from a wound. All bacteria were routinely grown in Todd-Hewitt broth (THB.sup.2, Difco, Detroit, DI, USA) and incubated at 37° C. in a humid atmosphere with 5% CO.sub.2.
[0203] Recombinant Expression and Purification of N- and C-Terminal VWA Domains of Collagen Type VI α-Chains
[0204] Collagen type VI microfibrils were extracted from bovine cornea by collagenase digestion as described by Abdillahi et al. (36). The cDNA constructs coding for the non-collagenous domains of collagen type VI were generated by RT-PCR on total RNA from mouse brain and cloned with 5′-terminal Nhel or Xhol and 3′-terminal BamHl or Xhol restriction sites using the following primers, see Table 2 (38).
TABLE-US-00003 TABLE 2 Primers and restriction enzymes used for RT-PCR analysis of globular regions of collagen type VI. Restriction SEQ Primer Sequence Enzyme ID NO: α1N(fw) 5′-AGAGCTAGCATGCCCTGTGGATCTATTC-3 Nhel 24 α1N(rev) 5′-GCACTCGAGAATCATGTCCACAATGGTGT-3′ Xhol 25 α1C(fw) 5′-GCAGCTAGCTGCACATGTGGACCCATTGA-3′ Nhel 26 α1C(rev) 5′-AACCTCGAGGCCCAGTGCCACCTTCCT-3′ Xhol 27 α2N(fw) 5′-AGAGCTAGCAAGGCCGACTGCCCAGTC-3′ Nhel 28 α2N(rev) 5′-GCACTCGAGGACCTTGATGATGCGGTT-3′ Xhol 29 α2C(fw) 5′-GAAGCTAGCTGTGAGAAGCGCTGTGGT-3′ Nhel 30 α2C(rev) 5′-GCAGGATCCACAGATCCAGCGGATG-3′ BamHl 31 α3N(fw) 5′-TATCTCGAGCTGATGGATCTGCTGTGAGGTTA-3′ Xhol 32 α3N(rev) 5′-AGGAACCAGGGATCCCAGGGGCCTGTCATACATGA BamHl 33 AGCC-3′ α3C(fw) 5′-AAAGCTAGCCTGGAGTGCCCTGTATTCCCAAC-3′ Nhel 34 α3C(rev) 5′-TTTGGATCCTCAAACTGTTAACTCAGGACTAC-3′ BamHl 35
[0205] Each of the amplified PCR products were inserted into a modified pCEP-Pu vector containing an N-terminal BM-40 signal peptide and a C-terminal tandem strepII-tag downstream of the restriction sites (39). HEK293-EBNA cells (Invitrogen, Carlsbad, Calif.) were transfected with the recombinant plasmids using FuGENE 6 reagent (Roche, Mannheim, Germany) according to the manufacturer's protocol. The cells were selected with puromycin (1 μg/ml) (Sigma-Aldrich, St. Louis, Mo.) and the recombinant proteins were purified directly from Dulbecco's modified eagle's medium (Invitrogen) supplemented with fetal calf serum (Biochrom GmbH, Berlin, Germany). After filtration and centrifugation (1 h, 10,000×g), the cell culture supernatants were applied to a Streptactin column (1.5 ml, IBA GmbH, Gottingen, Germany) and eluted with 2.5 mM desthiobiotin (Sigma-Aldrich), 10 mM Tris-HCl, pH 8.0.
[0206] Viable Count Assay
[0207] Bacteria were grown to mid-logarithmic phase (OD.sub.620≈0.4) in THB-medium at 37° C. with 5% CO.sub.2. The bacterial solution was subsequently washed and adjusted to 2×10.sup.9 cfu/ml in 10 mM Tris, pH 7.4, containing 5 mM glucose. S. pyogenes, S. aureus, E. coli or P. aeruginosa were then incubated with 2 μM of purified collagen type VI at 37° C. for 2 h. In some experiments S. pyogenes was incubated with recombinant collagen type VI fragments at various concentrations (0.125, 0.25, 0.5, 1.0 and 2.0 μM) for 2 h at 37° C. Bacteria incubated with Tris-HCl pH 7.4 buffer or 3 μM LL-37 (Innovagen, Lund, Sweden) were used as negative and positive controls respectively. To quantify the bactericidal activity, serial dilutions of the incubation mixtures were plated on blood agar plates, followed by incubation at 37° C. overnight, and the number of colony forming units (cfu) were determined. Hundred percent survival was defined as total survival of bacteria in the same buffer and under the same condition in the absence of collagen type VI or recombinant proteins.
[0208] Scanning Electron Microscopy
[0209] S. pyogenes, S. aureus, E. coli or P. aeruginosa (2×10.sup.9 cfu/ml) were incubated with purified collagen type VI at a concentration of 2 μM for 0, 30, 60 and 120 min at 37° C. with 5% CO.sub.2. In some experiments S. pyogenes was incubated with 2 μM of recombinant collagen type VI fragments for 2 h at 37° C. 3 μM of LL-37 was used as a positive control and bacteria in Tris-HCl, pH 7.4 was used as negative control. Samples were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4 (cacodylate buffer), washed with cacodylate buffer and dehydrated with an ascending ethanol series as previously described (40). The specimens were then subjected to critical-point drying with carbon dioxide and absolute ethanol was used as an intermediate solvent. The tissue samples were mounted on aluminium holders, sputtered with 20 nm palladium/gold, and examined in a Philips/FEI XL 30 FEG scanning electron microscope operated at 5 kV accelerating voltage.
[0210] Fluorescence Microscopy
[0211] Bacteria were grown to mid-logarithmic phase in THB-medium, washed and resuspended in 10 mM Tris-HCl containing 5 mM glucose to obtain a suspension of 2×10.sup.7 cfu/ml. 100 μl of the bacterial suspension was incubated with 2 μM of purified collagen type VI or 3 μM LL-37 at 37° C. for 30 min, followed by addition of 200 μl of FITC (6 μg/ml, Sigma-Aldrich) and incubated for 30 min at 37° C. Bacteria were washed and immobilized onto poly-L-lysine (Sigma-Aldrich) coated glass slides by incubating for 45 min at 37° C. The slides were washed with Tris-HCl/glucose and were fixes with 4% paraformaldehyde (PFA) by incubating at 4° C. for 15 min followed by 45 min incubation at RT. The glass slides were subsequently mounted on coverslips using Prolong Gold antifade reagent mounting medium (Invitrogen). The bacteria were visualized in a Nikon Eclipse E80i fluorescence microscope equipped with a Nikon DS-Fi 1 camera, a Plan Apochromat (100× objective) and a high numerical aperture oil condenser.
[0212] Heparin-Binding Assay
[0213] LL-37 (5 μg) or recombinant fragments from collagen type VI (10 μg) were applied to nitrocellulose membranes (Hybond-C; GE Healthcare, Uppsala, Sweden). Membranes were blocked with 2% BSA in PBS (w/v) for 2 h at RT, followed by washing steps with PBST (PBS with Tween-20) and incubated with 60 μg of heparin-biotin (Sigma-Aldrich) overnight at 4° C. In some experiments, unlabeled heparin (6 mg/ml) was added for competition of binding. After washing, the membranes were incubated with HRP streptavidin (Sigma-Aldrich) for 30 min at RT, washed and the bands were visualized by the Supersignal West Pico Chemi-luminescent substrate developing system (Thermo Fischer Scientific, Roskilde, Denmark).
[0214] Transmission Electron Microscopy
[0215] The binding of recombinant collagen type VI fragments to the bacterial surface was visualized by negative staining and transmission electron microscopy as described previously (35). Briefly, bacteria were incubated with recombinant collagen type VI fragments in presence or absence of heparin (10 μg/ml) for 1 h at 37° C. For visualization in the electron microscope the different recombinant fragments were conjugated with 5 nm colloidal gold (41). Specimens were examined in an Philips/FEICM 100 TWIN transmission electron microscope operated at 60 kV accelerating voltage. Images were recorded with a side-mounted Olympus Veleta camera and the ITEM acquisitions software.
[0216] Sequence and Structural Analysis
[0217] The amino acid sequence of human collagen type VI α-chains can be accessed through UniProtKB database; α1(VI) (UniProt # P12109), α2(VI) (UniProt # P12110) and α3(VI) (UniProt # P12111). Swiss PDB viewer DeepView version 4.1 was used for sequence alignment and to analyze three-dimensional structures. Only a crystal structure of mouse α3N5 was available with PDB code 4IGI (42). No crystal structures were available for human VWA domains of collagen type VI and predicted models from ModBase (modbase.compbio.ucsf.edu) were therefore used to generate the figures. There were no predicted models for N1 and C2 domains of α3(VI).
[0218] Peptide Synthesis
TABLE-US-00004 GVR28 [SEQ ID NO: 1] (GVRPDGFAHIRDFVSRIVRRLNIGPSKV), FYL25 [SEQ ID NO: 2] (FYLKTYRSQAPVLDAIRRLRLRGGS), FFL25 [SEQ ID NO: 3] (FFLKDFSTKRQIIDAINKVVYKGGR), VTT30 [SEQ ID NO: 4] (VTTEIRFADSKRKSVLLDKIKNLQVALTSK), SFV33 [SEQ ID NO: 5] (SFVARNTFKRVRNGFLMRKVAVFFSNTPTRASP), and DVN32 [SEQ ID NO: 6] (DVNVFAIGVEDADEGALKEIASEPLNMHMFNL)
[0219] were synthesized by Biopeptides (San Diego, Calif.). The purity (>95%) and molecular mass of these peptides was confirmed by MALDI-TOF MS analysis. All peptides used were water-soluble except DVN32, which was dissolved in <0.01% DMSO.
[0220] Radial Diffusion Assay
[0221] Radial diffusion assay (RDA).sup.2 was performed essentially as described earlier (43, which is incorporated herein by reference) with some minor modifications. Bacteria were grown to mid-logarithmic phase (OD.sub.620≈0.4) in 10 ml of full-strength (3% w/v) trypticase soy broth (TSB).sup.2 (Becton Dickinson, Franklin Lakes, N.J.). Bacteria were then washed once with 10 mM Tris-HCl (containing 5 mM glucose; pH 7.4). Subsequently, 4×10.sup.6 cfu/ml of bacteria were added to 5 ml of the underlay agarose gel, consisting of 0.03% (w/v) TSB, 1% (w/v) low electro endosmosis-type agarose and 0.02% (v/v) Tween 20 (both from Sigma-Aldrich). The underlay was poured into a Ø 90 mm Petri dish. After agarose solidification, Ø 4 mm wells were punched out, and 6 μl of 10 mM Tris-HCl buffer alone or containing peptide (100 μM) were added to each well. Plates were incubated at 37° C. for 3 h to allow diffusion of the peptides. The underlay gel was then covered with 5 ml of the overlay (6% TSB and 1% low electro endosmosis-type agarose in distilled H.sub.2O). Antimicrobial activity was seen as a clearing zone around each well after incubating 18-24 h at 37° C.
[0222] Statistical Analysis
[0223] Student's t test was performed to determine statistical significance. Values were expressed as means±standard errors and significance was determined as a P value of <0.05.
[0224] Results and Conclusions
[0225] Collagen Type VI Kills Gram-Positive and Gram-Negative Human Pathogens by Membrane Permeabilization
[0226] An integrated approach was established to combine microbiological and biochemical assays with high resolution scanning electron microscopy in order to investigate the antimicrobial properties of collagen type VI. Viable count assays were performed by incubating the Gram-positive bacteria S. pyogenes and S. aureus as well as the Gram-negative bacteria E. coli and P. aeruginosa with purified collagen type VI for 2 h at 37° C. The results showed that collagen type VI indeed displayed antibacterial activity against S. aureus, E. coli and P. aeruginosa in a similar way as observed for S. pyogenes, the chosen model organism (
[0227] The Recombinant VWA Domain-Containing Globular Regions of Collagen Type VI Bind to the Bacterial Surface in a Heparin-Dependent Manner
[0228] The affinity for negatively charged surfaces on bacterial membranes is a prerequisite for any given antimicrobial molecule for induction of bacterial killing, regardless of their mode of action. Thus, most antibacterial peptides and proteins are characterized by their affinity for heparin (32, 44). To determine whether collagen type VI exhibited similar properties biotin-labeled heparin was tested in a slot blot assay for binding to immobilized recombinant fragments of collagen type VI (as denoted in
[0229] The Recombinant VWA Domain-Containing Globular Regions of the Three Collagen Type VI α-Chains Induce Bacterial Killing by Membrane Disruption
[0230] In order to correlate the antibacterial activity of collagen type VI to individual N- and C-terminal globular regions, the bactericidal effect of the recombinant proteins on streptococci was investigated. Bacteria from the S. pyogenes strain AP1 were incubated with increasing concentrations of protein and analysed by viable count assays. The bacteria showed dose-dependent killing in the presence of the different fragments (
[0231] The VWA Domains of the Collagen Type VI α3-Chain Contain Amphipathic Amino Acid Motifs with Putative Antibacterial Activity
[0232] Cationic, hydrophobic and amphipathic properties are essential to the very core of antimicrobial peptide activity as the combination of these properties govern the extent to which bacterial killing is induced (9, 45). Therefore, in silico sequence analysis of the VWA domains of the α3(VI) chain was performed to identify such amino acid motifs with putative antimicrobial activity. The α3(VI) was chosen because it turned out to be most efficient in bacterial killing as described above. First, we defined the possible secondary structure by aligning the sequences of the N- and C-terminal VWA domains (N10-N2 and C1, see
[0233] Peptides Derived from the VWA Domains of the Collagen Type VI α3-Chain Exert Bactericidal Activity
[0234] Peptide sequences from putative antimicrobial regions with a high total net charge and hydrophobicity were identified, as these properties are important prerequisites for AMPs (46, 47). In total, five peptides were chosen from the N3, N2 and C1 domains (
Example B
[0235] Introduction
[0236] The purpose of this example was to further investigate the mode of action and immunomodulatory effects of host defence peptides derived from collagen type VI.
[0237] Materials and Methods
[0238] Bacterial Strains
[0239] Streptococcus pyogenes strain AP1 (40/58) of serotype M1 was from the World Health Organization Collaborating Centre for Reference and Research on Streptococci, Prague, Czech Republic. Staphylococcus aureus strain 111 and Escherichia coli strain B1351 were collected at the Department of Clinical Microbiology, Lund University Hospital, Sweden. The Pseudomonas aeruginosa strain used in this study was PAO1 (ATCC, Teddington Oly, UK), originally isolated from a wound.
[0240] Growth Media
[0241] All bacteria were routinely grown in Todd-Hewitt broth (THB, Difco, Detroit, DI, USA) by incubating at 37° C. in a humid atmosphere with 5% CO.sub.2.
[0242] Collagen Type VI Extraction and Peptide Synthesis Collagen type VI microfibrils were extracted from bovine cornea by collagenase digestion as described by Spissinger et al (34), with modifications from Bober et al. (35). In short, bovine corneas were cut into pieces and homogenized in Tris/saline buffer containing 5 mM calcium chloride and protease inhibitors. The homogenate was digested with collagenase type 1 (Worthington biochemical corporation, Lakewood, N.J.). Non-dissolved material was pelleted by centrifugation at 48,000×g for 20 min. The supernatant was applied in 500-μl aliquots onto a Superose 6 column of 25 ml (Amersham Biosciences, Uppsala, Sweden) equilibrated and eluted with homogenization buffer at 0.2 ml/minute. Fractions of 0.5 ml were collected, and those containing collagen VI were pooled and stored at 4° C. Collagen type VI-derived peptides (see Table 3) were synthesized by Biopeptides (San Diego, USA). LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) (SEQ ID NO: 36) was purchased (Innovagen AB, Lund, Sweden). The purity (>95%) and molecular mass of these peptides was confirmed by MALDI-TOF MS analysis. All peptides used were water-soluble except DVN32, which was dissolved in <0.01% DMSO (Sigma-Aldrich, St Louis).
[0243] Transmission Electron Microscopy
[0244] The binding of peptides to the surface of the bacteria and LPS was visualized by negative staining and transmission electron microscopy as described previously (35). Briefly, bacteria (2×10.sup.9 cfu/ml) were incubated with 2 μM peptide conjugated with 10 nm colloidal gold for 2 h at 37° C. with 5% CO.sub.2. For LPS (from Escherichia coli 0111:B4, Sigma-Aldrich) binding, 2 μM peptide conjugated with 10 nm colloidal gold was incubated with LPS (10 μg/ml) for 1 h at RT. Specimens were examined in an Philips/FEICM 100 TWIN transmission electron microscope operated at 60 kV accelerating voltage. Images were recorded with a side-mounted Olympus Veleta camera and the ITEM acquisitions software.
TABLE-US-00005 TABLE 3 Amino acid sequence and physiochemical properties of peptides used in this study. Amino acid MW Hydro- SEQ ID Peptide.sup.a sequence.sup.b (Da) Charge phobicity NO: GVR28 GVRPDGFAHIRDFVSRIV 3163 +4 46% 1 RRLNIGPSKV FYL25 FYLKTYRSQAPVLDAIRR 2937 +5 40% 2 LRLRGGS FFL25 FFLKDFSTKRQIIDAINK 2944 +4 40% 3 VVYKGGR VTT30 VTTEIRFADSKRKSVLLD 3403 +4 40% 4 KIKNLQVALTSK SFV33 SFVARNTFKRVRNGFLMR 3803 +7 48% 5 KVAVFFSNTPTRASP DVN32 DVNVFAIGVEDADEGALK 3490 −6 53% 6 EIASEPLNMHMFNL .sup.aPeptides are identified by their first three NH.sub.2-terminal residues using the single letter code, followed by the total number of residues constituting the peptide. .sup.bSequences of peptides are given in single letter code.
[0245] Circular Dichroism
[0246] Circular dichroism (CD) measurements were performed on a Jasco J-810 spectropolarimeter equipped with a Jasco CDF-4265 Peltier set to 25° C. Measurements were performed in at least duplicate in a 10-mm quartz cuvette under stirring with a peptide concentration of 30 mM. LPS (0.2 mg/ml) was added in some samples to study the effect on secondary structure of the peptides. This was monitored in the range of 200-260 nm (scan speed was 20 nm/min). Averages of five scans were baseline-subtracted.
[0247] Bactericidal Assay
[0248] Bacteria were grown to mid-logarithmic phase (OD.sub.620≈z 0.4) in THB medium at 37° C., with 5% CO.sub.2. The bacterial solution was subsequently washed and adjusted to 2×10.sup.7 cfu/ml in salt buffer (10 mM Tris-HCl, 150 mM NaCl supplemented with 5 mM glucose; pH 7.4) (both from Sigma-Aldrich). Various concentrations of peptides (0.3, 0.6, 3, 6, 30 and 60 μM) were incubated with bacteria in salt buffer with or without 20% human plasma. Bacteria incubated in only salt buffer with or without plasma were used as negative controls. Samples with LL-37 served as positive controls. The samples were incubated for 2 h at 37° C. with 5% CO.sub.2. To quantify the bactericidal activity, serial dilutions of the incubation mixtures were plated on THB agar plates, followed by incubation at 37° C. with 5% CO.sub.2 overnight, and the number of cfu were determined. Experiments were performed in triplicate. Hundred percent survival was defined as total survival of bacteria in the same buffer and under the same condition in the absence of peptides.
[0249] Propidium Iodide Uptake Assay
[0250] Bacterial membrane permeabilization was assessed by using propidium iodide (P1) (Sigma-Aldrich) dye as described previously (39). Briefly, bacteria were grown to mid-logarithmic phase (OD.sub.620≈0.4), washed and adjusted to 2×10.sup.9 cfu/ml. Bacteria (diluted 1:100) were mixed with peptides (30 μM final concentration) in the presence of salt buffer with or with plasma and incubated for 2 h at 37° C., with 5% CO.sub.2. PL (0.5 mg/ml) was added to each sample and incubated for 30 min on ice in darkness. Samples were analyzed on a Flow cytometer (BD Accuri flow cytometer, Becton Dickinson, Franklin Lakes). Bacteria incubated in only salt buffer with or without human plasma were used as negative controls. As a positive control, bacteria were treated with 70% ethanol for 20 min at room temperature. The percentage of membrane permeabilization was calculated as the percent of fluorescent intensity of peptide-treated samples with respect to fluorescence intensity of untreated samples.
[0251] Scanning Electron Microscopy
[0252] Bacteria (2×10.sup.9 cfu/ml) were incubated with 30 μM peptide for 2 h at 37° C. under physiological conditions such as salt buffer with or without plasma. Samples were fixed with cacodylate buffer (2.5% glutaraldehyde (Merck, Germany) in 0.1 M sodium cacodylate (Sigma-Aldrich), pH 7.4, washed with cacodylate buffer and dehydrated with an ascending ethanol series as previously described (40). The tissue samples were mounted on aluminium holders, sputtered with 20 nm palladium/gold, and examined in a Philips/FEI XL 30 FEG scanning electron microscope operated at 5 kV accelerating voltage.
[0253] Membrane Permeabilization Assay
[0254] Dry lipid films of E. coli polar lipid extract were formed in round-bottom flask walls by dissolving lipids in chloroform, followed by evaporation under N.sub.2-flow and subsequently placed in vacuum overnight. Lipid films were re-suspended either by 30 min stirring (E. coli), at 55° C. in an aqueous solution of 100 mM 5(6)-carboxyfluorescein in 10 mM Tris (set to pH 7.4 at 37° C.). Suspensions were then vortexed followed by repeated extrusion through a 100 nm polycarbonate membrane mounted in a LipoFast mini-extruder (Avanti Polar Lipids) in order to reduce multilamellar structures and polydispersity. Un-trapped carboxyfluorescein was removed by gel filtration on Sephadex PD-10 columns (GE Healthcare, Little Chalfont, UK). Membrane permeability was measured by monitoring carboxyfluorescein efflux from the liposomes to the external low concentration environment, resulting in loss of self-quenching and an increased fluorescence signal with excitation and emission wavelengths of 492 and 517 nm, respectively. Fluorescence was measured with a Varioskan Flash Multimode Reader (Thermo Fisher Scientific, Waltham, Mass.) in black Nunc Delta Surface 96-well plate (Thermo Fisher Scientific, Roskilde, DK). The wells were prepared with a 2-fold serial dilution of the peptides in tris buffer, as well as controls without peptides (background) and 0.16% Triton X-100 (maximum leakage). The plates were pre-heated to incubation temperature (37° C.) and administered liposome solution, to a final lipid concentration of 10 μM in 200 μl, with the Varioskan integrated dispenser. The effects of each peptide concentration on the liposome systems were monitored for 45 min, at which point the initial leakage had largely subsided. Results shown represent the average from triplicate experiments with standard deviations and are expressed as percent of total leakage generated with Triton X-100 and subtraction of the baseline value. The EC.sub.50-values are calculated from a sigmoidal dose-response curve fitting with variable slope to the leakage percentage as a function of the peptide concentration (log 10), using Graphpad Prism.
[0255] Hemolysis Assay
[0256] Blood from healthy individuals was drawn into Vacutainer tubes (Becton Dickinson) containing EDTA and centrifuged at 800×g for 10 min. The plasma and buffy coat were removed. Erythrocytes were washed three times and resuspended in phosphate buffered saline (PBS, Medicago, USA). The cells were incubated with peptides (final concentration of 30 and 60 μM) for 1 h at 37° C. in end-over-end rotation. Cells incubated with 2% Triton X-100 (Sigma-Aldrich) served as positive control. The samples were then centrifuged at 800×g for 10 min. The supernatant was collected and the absorbance of hemoglobin release was measured at 540 nm and is expressed as % of Triton X-100 induced hemolysis.
[0257] Lactate Dehydrogenase (LDH) Assay
[0258] The cytotoxicity experiments were performed as described previously (41). Briefly, human monocytic THP-1 cells (American Type Culture Collection (ATCC), Manassas, Va.) were cultured into 96-well plates using Dulbecco's Modified Eagle's medium (DMEM) (PAA Laboratories) supplemented with 10% fetal calf serum. The medium was removed and the cells were subsequently washed with DMEM. Peptides (1, 5, 10, 20, 50 μM) diluted in DMEM were added in triplicates. The LDH based TOX-7 kit (Sigma-Aldrich) was used according to the manufacturer instructions. The amount of LDH release from dead cells was measured at 450 nm. As a positive control 2% Triton X-100 was used.
[0259] LPS Stimulation of Macrophages In Vitro
[0260] Murine macrophage-like cells (RAW 264.7; ATCC) were seeded in 96-well plates at 3.5×10.sup.5 cells/well in DMEM (without phenol red, PAA Laboratories) supplemented with 10% fetal calf serum and antibiotic-antimycotic (Invitrogen, Carlsbad, Calif.). After overnight incubation at 37° C., the cells were washed once with DMEM. LPS (10 ng/ml) was pre-incubated with the peptides for 20 min at room temperature and added to the cells. The cells were subsequently incubated for 24 h at 37° C. Griess reagent (Sigma, St. Louis, Mo.) was added to culture supernatant in 1:1 ratio followed by 15 min incubation in dark. The absorbance was then measured at 550 nm with a spectrophotometer. The cells with and without LPS stimulation were taken as positive and negative controls for LPS-induction. For the determination of NO production with peptides and LPS treatments, the assay was done in triplicate and the average values were considered for each set. Cells were grown at 37° C. and with 5% CO.sub.2 in fully humidified air.
[0261] In Vitro Wound Healing Assay
[0262] HaCaT cells (ATCC) were cultured into a 24-well plate at 3×10.sup.5 cells/well with keratinocyte basal medium, (KBM Gold, Lonza Group AG, Switzerland) according to the manufacturer instructions, until confluence. Prior to experiment cells were cultured in serum-free medium for 24 h. The cell monolayer was subjected to a mechanical scratch wound using a sterile pipette tip. Detached cells were removed by washing twice with PBS. Collagen type VI-derived peptides (10 μg/ml), LL-37 (10 μg/ml) and collagen type VI (10 μg/ml) was added to the cells and incubated for 24 h at 37° C. Cells with and without the addition of 10% FCS in the basal medium were used as positive and negative controls, respectively. All experiments were performed in a humidified atmosphere with 5% CO.sub.2 at 37° C. Images of the wounded cell monolayer were taken using a microscope (Olympus, SC30 digital camera, Tokyo, Japan) at 0 and 24 h after scratched wounding.
[0263] Statistical Analysis
[0264] The data was analysed with Graphpad Prism 6. Student's t test was performed to determine statistical significance. Values were expressed as means±standard errors and significance was determined as a p value of <0.05.
[0265] Results and Conclusions
[0266] Collagen Type VI-Derived Peptides Adherence to Bacterial Surface
[0267] To examine whether collagen type VI-derived peptides (see in Table 3) interacts with bacterial surfaces, gold labelled peptides were incubated with P. aeruginosa and S. aureus and subjected to negative staining transmission electron microscopy. The electron micrographs revealed that SFV33 and LL-37 were able to adhere to the bacterial surface of S. aureus and P. aeruginosa (
[0268] Binding of Collagen Type VI-Derived Peptides to LPS
[0269] In the next series of experiments, to determine whether Lipopolysaccharide (LPS) of Gram-negative bacteria could serve as a potential target for collagen type VI-derived peptides, E. coli LPS was incubated with gold conjugated peptides for 1 h and subjected to negative staining transmission electron microscopy. Both LL-37 and SFV33 bound to LPS (
[0270] Collagen type VI-derived peptides show antibacterial activity at physiological conditions The antibacterial activities of many AMPs are inhibited in a physiological environment such as high salt concentration or the presence of plasma proteins (48, 49). Viable count assays were performed using collagen type VI derived peptides. For this purpose, a panel of Gram-positive bacteria S. pyogenes and S. aureus as well as the Gram-negative bacteria E. coli and P. aeruginosa were subjected to collagen type VI-derived peptides in the presence of physiological salt with or without 20% human plasma. For comparison, the classical AMP LL-37 was used at the same concentrations.
[0271] Membrane-Permeabilizing Activity of Collagen Type VI-Derived Peptides
[0272] In order to investigate the effects of collagen type VI-derived peptides on bacterial membranes, propidium iodide uptake was measured. As shown in
[0273] The effect of SFV33 on bacterial membranes was further examined with high resolution scanning electron microscopy. At the ultrastructural level, SFV33 caused disruption of bacteria, leading to disintegration and ejection of cytoplasmic components in the presence of salt (P. aeruginosa and S. aureus) and plasma (P. aeruginosa) (
[0274] To investigate the effect of collagen type VI-derived peptides on membranes a liposome model was used to study membrane permeabilization. Peptides were tested for membrane leakage in a liposome model membrane system (E. coli polar lipid extract). The results showed that all peptides have the ability to cause membrane permeabilization at physiological pH, except DVN32 (
[0275] Peptides Effect on Eukaryotic Cells
[0276] One major side effect for some AMPs is that they do not only act on bacterial membranes but they can also destroy and eliminate eukaryotic cells. The cytotoxic effect of different concentrations of peptides on erythrocytes and monocytes was assessed. 2% Triton X-100 was used as cytotoxic agent, a positive control. The results show that peptides did not show any toxicity towards erythrocytes and monocytes at concentrations up to 30 μM, in contrast to LL-37 (
[0277] Immunomodulatory Properties of Collagen Type VI-Derived Peptides
[0278] LPS is a well-studied endotoxin released by the outer membrane of Gram-negative bacteria and play an important role in pathogenesis of certain bacterial diseases. A massive release of LPS can cause endotoxic shock, and lead to death (50). The immunomodulatory effects of collagen type VI derived peptides were investigated. Murine macrophage-like cells were stimulated simultaneously with E. coli LPS and collagen type VI-derived peptides and the amount of nitrite in the supernatant was measured with Greiss reagent. As shown in
[0279] To examine the biological effect of synthetic collagen type VI-derived peptides on wound healing, HaCaT cells were cultured in wells of 24-well plate; cells were scratched and incubated with intact collagen type VI, collagen type VI-derived peptides or LL-37 (10 μg/ml). Cell migration was recorded by photomicrograph at post-scratched 0 hour and 24 h. As illustrated in
Example C
[0280] Introduction
[0281] The purpose of this study was to assess the antimicrobial effects of collagen type VI derived peptides on bacteria which are resistant to many conventional antibiotic agents (52, 53). Additionally, this study assessed the effect of collagen type VI on the membranes of resistant bacteria.
[0282] Materials and Methods
[0283] Microorganisms and Culture Conditions
[0284] The following multidrug-resistant bacterial strains were kindly provided by Lisa Påhlman (Dept. of Infection Medicine, Lund University): Pseudomonas aeruginosa (MRPA), Staphylococcus aureus (M RSA) Escherichia coli (MREC), Staphylococcus epidermidis (MRSE) and Klebsiella pneumoniae (MRKP) All tested multidrug-resistant microorganisms were clinical isolates from patients with either bacteremia or pneumonia. All strains were grown overnight in Todd-Hewitt broth (THB, Gibco, Grand Island, N.Y. USA) at 37° C. in a humid atmosphere containing 5%.
[0285] Antibacterial Activity Assay
[0286] Bacteria were grown to mid logarithmic phase in Todd-Hewitt broth (OD.sub.620≈0.4), harvested by centrifugation at 3,500 rpm for 10 min and washed twice in TBS buffer. Bacterial suspensions were adjusted to 2×10.sup.9 colony forming units (cfu) per ml. The bacteria were further diluted in TBS and incubated with different collagen type VI or collagen type VI peptide concentrations. Bacteria incubated with TBS or 3 μM LL-37 antimicrobial peptide (Innovagen, Lund, Sweden) were used as negative or positive controls, respectively. Samples were incubated for 2 h at 37° C. in a humid atmosphere containing 5% CO.sub.2. Serial dilutions were plated on agar plates, incubated overnight at 37° C., and the number of cfu were thereafter determined by counting visible colonies. Experiments were performed in triplicate.
[0287] Scanning Electron Microscopy
[0288] For high resolution field emission transmission electron microscopy (FESEM), specimens were fixed over night at RT with 2.5% glutaraldehyde in cacodylate buffer. They were then washed with cacodylate buffer and dehydrated with an ascending ethanol series from 50% (v/v) to absolute ethanol. The specimens were then subjected to critical point drying with carbon dioxide and absolute ethanol was used as an intermediate solvent. The tissue samples were mounted on aluminum holders, sputtered with 20 nm palladium/gold, and examined in a Philips/FEI XL 30 FESEM scanning electron microscope using an Everhart-Tornley secondary electron detector.
[0289] Results and Conclusions
[0290] Collagen Type VI and Collagen Type VI-Derived Peptides are Antimicrobial Against Multidrug-Resistant Mammalian Pathogens
[0291] In order to assess possible antibacterial effects of collagen type VI and peptides thereof, bacteria were treated with purified preparations of this protein and its peptides. Bacteria treated with TBS buffer or the cathelicidin peptide LL-37 served as negative and positive controls, respectively. The results from viable-count assays show killing of multidrug-resistant human pathogens (
[0292] Collagen Type VI Killing Properties are Associated with Streptococcus and Pseudomonas Membrane Disruption as Determined by Electron Microscopy—
[0293] For a more detailed understanding of the underlying killing mechanism human skin biopsies were inoculated with MRSA and MRPA (as model systems) in the presence or absence of collagen type VI and visualized by scanning electron microscopy.
Example D
[0294] Introduction
[0295] The purpose of this study was to assess the antimicrobial effects of amphipathic peptides derived from the collagen VI amino acid sequence, in an in vivo infection model in mice, of bacterial infection with an invasive Pseudomonas aeruginosa (abbreviated as P. aeruginosa).
[0296] Materials and Methods
[0297] Microorganisms and Culture Conditions
[0298] P. aeruginosa 15159 strain was grown overnight in Todd-Hewitt broth (THB, Gibco, Grand Island, N.Y. USA) at 37° C. in a humid atmosphere containing 5% CO.sub.2.
[0299] Animal Experiments
[0300] Animal experiments were performed according to a protocol approved by the Local Ethics Committee at Lund University. Animals were housed under standard conditions of light and temperature and had free access to standard laboratory chow and water. P. aeruginosa 15159 bacteria were grown to mid-exponential phase (A.sub.620˜0.5), washed and diluted in PBS to 2×10.sup.8 cfu/ml, and kept on ice until injection. Female BALB/c mice, 8 weeks old, were anesthetized with isoflurane and injected intraperitoneally with 100 μL of the bacterial solution, followed by injection of 100 μL SFV33 or GVR28 peptide (1 mg/mL) after 15 minutes, or with 100 μL PBS alone (control group).
[0301] Results
[0302] The Effect of Treatment with Collagen VI-Derived Peptides on Survival of Infected Mice
[0303] A beneficial effect of SFV33 and GVR28 was demonstrated in murine survival studies. Intraperitoneally infected mice were treated with a single dose of SFV33 or GVR28 15 minutes after infection, and the survival was recorded.
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